Since some substances belonging to hydroxycarboxylic acids are useful as a raw material for polymers or an intermediate for medicines, a process for effectively producing such substances have been in demand.
As an example, glycolic acid (α-hydroxyacetic acid) can be cited. Glycolic acid has been used as a cleaning agent or a raw material for cosmetics, but has recently been paid attention to as a raw material of polyglycolic acid which is useful as a gas barrier polymer or a medical polymer. The reason why glycolic acid has been paid attention to as a gas barrier material is that the layer of polyglycolic acid has the property of a high oxygen barrier and is provided with performance as a material for packing food or carbonated beverage which goes easily bad in the presence of oxygen.
In order to actually produce polyglycolic acids on the industrial scale in the future, glycolic acid that is its raw material must be supplied in high purity and at low cost. However, glycolic acid of a chemically synthesized product which is currently available from the market contains not a few impurity contents, so there is a problem as a raw material for polymers in terms of purity. It is because these impurity contents not only prevent a dehydrative condensation reaction of glycolic acid, but also methoxy acetate that is one of these impurity contents is a compound suspicious of carcinogenic potential. Therefore, it is not desirable that such impurity contents are included in a packing material for food or beverage. Of course, it is technically possible to remove impurity contents by purification, but such purified products actually involve high cost. Thus, such purified products are not realistic as a raw material for packing at low cost.
In order to avoid the aforementioned problem shown in glycolic acid of the chemically synthesized product, production of glycolic acid using ethylene glycol (hereinafter may be referred to as EG) as a raw material has been attempted according to the biological method. In Japanese Patent Laid-open Nos. H10-174593 (1998-174593) and H10-174594 (1998-174594), there has been disclosed a method for producing glycolic acid by a microorganism, which comprises culturing yeast belonging to Pichia (genus), Rhodotorula (genus), Sporobolomyces (genus), Kluyveromyces (genus) or Torulopsis (genus), a strain belonging to Nocardia (genus), a strain belonging to Rhodococcus (genus), or an Escherichia coli B strain in a medium containing ethylene glycol for separating and collecting glycolic acid from the culture broth. Incidentally, there has been disclosed that the yield of glycolic acid is low relative to Escherichia coli K12 strain. Of the methods for producing glycolic acid as described in Examples of Japanese Patent Laid-open Nos. H10-174593 (1998-174593) and H10-174594 (1998-174594), a method comprising employing Pichia naganishii results in the highest accumulation concentration of glycolic acid, obtaining 35.3 g/L of glycolic acid in a reaction for 30 hours. The production of glycolic acid employing Pichia naganishii has been reported in a literature (Kataoka, M., et al., Biosci. Biotechnol. Biochem., Vol. 65 (10) pp. 2265-2270 (2001)) that 105 g/L of glycolic acid is obtained by a reaction for 120 hours due to further improvement in the reaction conditions. In short, in a method for producing glycolic acid employing Pichia naganishii, the time required for a reaction from ethylene glycol to glycolic acid is long, that is 120 hours, which causes an increase in the production cost of glycolic acid. So, the reaction time has been required to be reduced when the actual production on the industrial scale is supposed. And there has been a problem such that, in this production method, by-product organic acids produced by a microorganism in use are entrained, thus causing difficulties in a purification process or a dehydrative condensation reaction thereafter.
Regarding a metabolic reaction from ethylene glycol to glycolic acid by a microorganism, the results of study using Escherichia coli conducted by Boronat et al. have been disclosed (Boronat, A., et al., J. Bacteriol., Vol. 153 (1), pp. 134-139, (1983)), namely, a two-stage metabolic pathway including a reaction from ethylene glycol to glycolaldehyde and a reaction from glycolaldehyde to glycolic acid. Boronat et al. have paid attention to the fact that propanediol oxidoreductase (hereinafter may be referred to as PDO redox enzyme) of a catalytic enzyme of a reaction from 1,2-propanediol (hereinafter may be referred to as PDO) to lactaldehyde also recognizes ethylene glycol as a substrate (Boronat, A., et al., Biochim. Biophys. Acta, Vol. 672, pp. 98-107, (1981)). A strain which has the enhanced activity of glycolaldehyde dehydrogenase (hereinafter may be referred to as GAL dehydrogenase) of a catalytic enzyme of a reaction from glycolaldehyde to glycolic acid is isolated from strains in which activity of the PDO redox enzyme is enhanced by mutation. Incidentally, Table 1 below is cited from a thesis by Boronat et al.
TABLE 1Activity ofActivity ofPDO redoxGALenzymedehydrogenasePDOEG(U/mg(U/mgutilizingutilizingStrainsprotein)protein)capabilitycapabilityG1 strain0.0050.145NoNo(referencedstrain)G3 strain0.1300.165YesNoEG3 strain0.4150.560(notYesmentioned)
An Escherichia coli G1 strain of the referenced strain is able to utilize neither of 1,2-propanediol nor ethylene glycol. A G3 strain that is a mutant strain in which activity of the PDO redox enzyme is remarkably enhanced has acquired the PDO utilizing capability, but still cannot be able to utilize EG. On the basis of these results, Boronat et al. have reasoned that the reason why the G3 strain cannot be able to utilize EG might be that a reaction from glycolaldehyde to glycolic acid does not proceed due to insufficient activity of GAL dehydrogenase. They have concluded that their reasoning is right because a mutant EG3 strain in which activity of GAL dehydrogenase is remarkably enhanced is isolated, and this strain utilizes EG. In other words, Boronat et al. insist that activity of GAL dehydrogenase must be sufficient in order to produce glycolic acid from EG.
On the other hand, a new knowledge disclosed in the present specification is that enhancement of GAL dehydrogenase activity is not necessarily required in order to produce glycolic acid from EG, but only enhancement of activity of PDO redox enzyme may be sufficient. Such a difference is caused because Boronat et al. have only paid attention to enhancement of GAL dehydrogenase activity in the EG3 strain and have not paid attention to enhancement of activity of PDO redox enzyme as well at the same time. Considering the test results of Boronat et al. again based on our knowledge, the EG3 strain is able to acquire the EG utilizing capability not because activity of GAL dehydrogenase is enhanced, but because activity of PDO redox enzyme is enhanced.
Furthermore, according to a report by Boronat et al., in the EG3 strain, glycolaldehyde that is an intermediate is accumulated under the condition in the presence of glycolic acid along with EG as a substrate. Namely, this shows to cause troubles such that glycolaldehyde that is an intermediate is accumulated as impurity content when glycolic acid is produced from EG as a raw material according to the biological method using Escherichia coli. 
According to the past knowledge from the aforementioned facts, it is shown that enhancement of activity of GAL dehydrogenase is absolutely necessary when glycolic acid is produced from EG using Escherichia coli and glycolaldehyde that is an intermediate is accumulated in the produced glycolic acid. From such a reason, even the skilled person in the art wasn't able to easily suppose that glycolic acid can be selectively produced without enhancement of GAL dehydrogenase activity and glycolic acid can be even produced with good efficiency without including glycolaldehyde of an intermediate.
Various investigations of methods for producing glyceric acid as example of hydroxycarboxylic acids besides glycolic acid have also been performed. In such investigations, as a method for producing glyceric acid using inexpensive glycerol as a raw material, a chemical synthesis method comprising using a Pt catalyst has been disclosed in Japanese Patent Laid-open No. H5-331100 (1993-331100) and a biological method by a microorganism belonging to Gluconobacter (genus) has been disclosed in Japanese Patent Laid-open No. H1-168292 (1989-168292). In the former method comprising using a Pt catalyst, the reaction selectivity of about 80% is never sufficient, thus generating by-products in a large amount and there is shown about a need to more strictly control the reaction temperature. In the latter biological method, a method for producing D-glyceric acid using glycerine as a raw material has been described, but there is a problem in that it takes a long time of 4 days required for the preparation of a microbial mass and the reaction and it is possible to easily suppose that a large amount of by-product organic acids derived from a microorganism in use are entrained into the prepared glyceric acid. By the way, there has not been known about a method for producing L-glyceric acid.
Furthermore, as an example of hydroxycarboxylic acid, a method for producing hydroxyethoxy acetate according to the biological method has been disclosed in Japanese Patent Laid-open No. S59-85296 (1984-85296). The method comprises culturing yeast belonging to Candida (genus), Torulopsis (genus), Rhodotorula (genus), Hansenula (genus), Debaryomyces (genus), Cryptococcus (genus) and Pichia (genus) in a medium containing diethylene glycol, separating and collecting hydroxyethoxy acetate from the culture broth. In this production method, it is shown that it takes a long time required for culturing and diglycolic acid in a large amount is also produced as a by-product. From the fact, it is easily imagined that great efforts for the separation and purification of hydroxyethoxy acetate are needed.    Patent Document 1: Japanese Patent Laid-open No. H10-174593 (1998-174593)    Patent Document 2: Japanese Patent Laid-open No. H10-174594 (1998-174594)    Patent Document 3: Japanese Patent Laid-open No. H5-331100 (1993-331100)    Patent Document 4: Japanese Patent Laid-open No. H1-168292 (1989-168292)    Patent Document 5: Japanese Patent Laid-open No. S59-85296 (1984-85296)    Non-patent Document 1: Kataoka, M., et al., Biosci. Biotechnol. Biochem., Vol. 65 (10), pp. 2265-2270, (2001)    Non-patent Document 2: Boronat, A., et al., J. Bacteriol., Vol. 153 (1), pp. 134-139, (1983)    Non-patent Document 3: Boronat, A., et al., Biochim. Biophys. Acta, Vol. 672, pp. 98-107, (1981)